Enzymatic thiolation of E. coli sRNA.

Abstract

Transfer RNA (sRNA'), which contains several unusual constituents such as pseudouridine,2 methylated bases,3' 4 dihydrouridine,' and inosine,6 has recently been found to contain still another class of minor components, the thionucleotides. These include 4-thiouridine7 and a 2-thiopyrimidine,8 although there are more members of this class as yet unidentified.9 In the case of the methylated bases, the methyl groups have been found to arise as additions to the four common bases in the polynucleotide chain of an sRNA precursor. 10 The present work indicates that the sulfur moieties may also be introduced at the macromolecular level. Studies with growing cells using the isotope dilution technique have shown that cysteine or cystine, of all the common sulfur intermediates tested, are the most direct donors of the sulfur atom of thionucleotides."1 Evidence is presented here that in a cell-free system, S35-cystine can donate its sulfur atom to E. coli sRNA to form S35-labeled thionucleotides. Some cofactor and acceptor requirements of the reaction are outlined. In addition, data are presented to show that the acceptor pyrimidine of sRNA which results in the formation of 4-thioUMP is a uracil unit. Materials and Methods.-Yeast and E. coli B sRNA were obtained from General Biochemicals, Inc., and further purified by phenol extraction and dialysis. Rabbit liver sRNA was prepared by the method of Cantoni et al.'2 C14-cytidine, uniformly labeled, was obtained from Schwarz Bio-Research, S'5-cystine from Amersham and from Schwarz BioResearch, and S"-H2SO4 from New England Nuclear Corp. Alcohol-resistant membrane filters (Metricel type GA-6) were obtained from Gelman Instrument Company, Ann Arbor, Michigan. The sulfur-poor sRNA was prepared using mid-log phase cells of E. coli 58-161 (met-), a methionine auxotroph showing "relaxed control" of nucleic acid synthesis. The cells were grown in a minimal medium" containing 2 X 10-5 M sulfate and 4.2 X 10-1 M methionine. The sRNA was prepared by the method of Fleissner and Borek,'0 and showed an absorbance at 335 mu equal to 0.76 for each 100 A260 units, as compared to a normal level of 1.5-1.7 for E. coli B. Ribosomal RNA was prepared from a sample of ribosomes from E. coli K12S obtained from Dr. A. Weissbach. The ribosomal pellet from a 105,000 X g centrifugation was washed with water to dissociate polysomes and remove any last traces of sRNA before recentrifuging at 105,000 X g for 3 hr. The pellet was taken up in 0.01 M Tris buffer, pH 7.4, and shaken with an equal volume of 90% phenol. The aqueous supernatant was combined with a buffer washing of the phenol layer and precipitated with 2 vol of cold ethanol. The RNA precipitate was dialyzed overnight against distilled water. A spectrum of the material gave no evidence for an absorption peak at 335 mu.

Mammalian tRNA sulfurtransferase: properties of the enzyme in rat liver

“…The required components of the reaction are enzyme, tRNA, Mg , ATP, and cysteine. In this regard, the requirements are similar to those reported previously for E. coli sulfurtransferases (11,12,14).…”

“…Only (11,14,20) and rat brain (15) also contain thionucleotides which chromatograph at this position on DEAE-cellulose. We are currently evaluating the isolated hepatocyte system (25) and certain cell lines maintained in culture to see if [35 ] tRNA can be isolated for use as an in vivo marker, and an aid to identification of the presumed in vitro labeled thionucleotide.…”

“…The latter enzyme has been fractionated into two components, one which requires ATP, a divalent metal ion, tRNA and a sulfhydryl compound, and the other requires cysteine as sulfur donor and the product of the first enzyme. The enzymes of Bacillus subtilis have been studied (13), and differ from the enzymes of E. coli in the sulfur donor utilized and the thionucleotides formed in vitro (11,12). It was also found that the B. subtilis sulfurtransferases were more efficient with P-mercaptopyruvate than cysteine as substrate, while the E. coli enzyme was not active with the former sulfur donor.…”

“…Several reports of RNA sulfurtransferase in bacteria have appeared (11)(12)(13)(14), but only the s4U:sulfurtransferase (2) of Escherichia coli has been purified (14). The latter enzyme has been fractionated into two components, one which requires ATP, a divalent metal ion, tRNA and a sulfhydryl compound, and the other requires cysteine as sulfur donor and the product of the first enzyme.…”

MiaB Protein Is a Bifunctional Radical-S-Adenosylmethionine Enzyme Involved in Thiolation and Methylation of tRNA

“…Cells growing in the presence of [ 35 S]cysteine generate 35 S-labeled ms 2 i 6 A demonstrating that the primary source of the sulfur atom in ms 2 i 6 A is cysteine (36,37). However, cysteine is certainly not the ultimate sulfur donor, and the question is how sulfur is mobilized from cysteine.…”